Particle-nucleic acid conjugates and therapeutic uses related thereto

ABSTRACT

This disclosure relates to particles conjugated to therapeutic nucleic acids. In certain embodiments, the nucleic acid comprises a sequence that catalytically cleaves RNA, e.g., DNAzyme or RNAzyme. In certain embodiments, the particles contain nucleic acids with both DNAzyme and/or RNAzyme and siRNA sequences. The cleaving nucleic acids optionally comprise a sequence functioning to hybridize to a target of interest and/or the particles are further conjugated to a targeting moiety. In certain embodiments, conjugated particles are used in the treatment or prevention of cancer or viral infections or bacterial infections. In certain embodiments, conjugated particles are used in detecting metal ions and other small molecule analytes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application61/663,933 filed Jun. 25, 2012 and is hereby incorporated by referencein its entirety.

FIELD

This disclosure relates to particles conjugated to therapeutic nucleicacids. In certain embodiments, the nucleic acid comprises a sequencethat catalytically cleaves RNA, e.g., DNAzyme or RNAzyme. In certainembodiments, the particles contain nucleic acids with both DNAzymeand/or RNAzyme and siRNA sequences. The cleaving nucleic acidsoptionally comprise a sequence functioning to hybridize to a target ofinterest and/or the particles are further conjugated to a targetingmoiety. In certain embodiments, conjugated particles are used in thetreatment or prevention of cancer or viral infections or bacterialinfections. In certain embodiments, conjugated particles are used indetecting metal ions and other small molecule analytes.

BACKGROUND

Nucleic acids such as siRNA and DNAzymes have been contemplated asactive ingredients in therapeutic strategies. DNAzymes are shortdeoxyribonucleotide sequences that cleave target RNA sequences, e.g.,mRNA. Small interfering RNA (siRNA) contains RNA sequences which aretypically between 20-25 nucleotides in length. siRNA interferes with theexpression of a specific gene containing the siRNA sequences. DNAzymeshold potential advantages over siRNA for therapeutic gene regulation dueto their innate ability to catalytically cleave mRNA without the needfor hijacking the RISC(RNA-induced Silencing Complex) machinery of thecell.

DNAzymes targeting mRNA of integrins reduced protein expression inendothelial cells and thus blocked microvascular endothelial cellcapillary tube formation. See e.g., Niewiarowska et al., Cancer GeneTher. 2009, 16(9):713-22. DNAzyme regulation of the EGFR gene expressionlevels was shown to inhibit the growth of cancer cells. See Santiago etal., Nature Med. 1999, 5:1264-9. DNAzymes that cleave VEGFR2 mRNA wereshown to limit the proliferation of endothelial cells and blocked tumorgrowth in vivo. Zhang et al., Cancer Res. 2002, 62:5463-9.

The movement of nucleic acids to a site of interest within a cellpresents a challenge to using nucleic acids as therapeutic agents. Cellmembranes generally prevent nucleic acids from migrating in and out ofcellular compartments. Cationic polymers or liposomes may be employed toimprove delivery. Cationic liposomes are toxic to cells. Once within thecells, biological processes degrade oligonucleotides. Chemicallymodifying the oligonucleotide backbone can slow nuclease activity. Inany case, there is exists a need to identify improved compositions andmethods.

Liu & Lu disclose a gold nanoparticle/DNAzyme assembly for a biosensorapplication. See Chem. Mater., 2004, 16, 3231-3238. See also Yang etal., Chem Commun, 2010, 46, 3107-3109. Rosi et al., discloseoligonucleotide-modified gold nanoparticles for intracellular generegulation. See Science 2006, 312, 1027. Giljohann et al., disclose generegulation with polyvalent siRNA-nanoparticle conjugates. See J. Am.Chem. Soc. 2009, 131, 2072. See also Hurst et al., Anal. Chem., 2006, 78(24):8313-8318 and Liu et al., Analyst, 2012, 137, 70-72. Referencescited herein are not an admission of prior art.

SUMMARY

This disclosure relates to particles conjugated to therapeutic nucleicacids. In certain embodiments, the nucleic acid comprises a sequencethat catalytically cleaves RNA, e.g., DNAzyme or RNAzyme. In certainembodiments, the particles contain nucleic acids with both DNAzymeand/or RNAzyme and siRNA sequences. The cleaving nucleic acidsoptionally comprise a sequence functioning to hybridize to a target ofinterest and/or the particles are further conjugated to a targetingmoiety. In certain embodiments, conjugated particles are used in thetreatment or prevention of cancer or viral infections or bacterialinfections. In certain embodiments, conjugated particles are used indetecting metal ions and other small molecule analytes.

In certain embodiments, the diameter of the particle is about 500 nm to5 nm or 200 nm to 10 nm or 5 nm. In certain embodiments, the particlecomprises or consists essentially of a metal such as gold, silver, iron,or iron oxide.

In certain embodiment, the disclosure contemplates cleaving nucleicacids which are DNAzymes that cleave RNA, e.g. mRNA or viral orbacterial RNA or mRNA. In certain embodiments, the DNAzyme may cleaveRNA independent of metal cations. In certain embodiments, the cleavingnucleic acid is selected from DNAzyme 10-23, DNAzyme 20-49, and DNAzyme8-17 or variants or modified forms thereof such as amine or imidazolylmodified deoxyadenosines. In certain embodiments, the cleaving nucleicacid is DNAzyme 10-23 coating the particle at about one nucleic acid toabout 3 to 12 square nanometers of the particle surface. In certainembodiments, the cleaving nucleic acid is DNAzyme 10-23 coating theparticle at about or greater than 8, 9, 10, or 11 square nanometers pernucleic acid or less than 12 or 15 square nanometers per nucleic acid.

In certain embodiments, the particle is conjugated to the nucleic acidthrough a linking group comprising a thiol group, metal ligand, ethyleneglycol polymer, alkyl chain, ester group, or amide group. In certainembodiments, a metal particle is coated with a polymer and the nucleicacid is conjugated to the polymer. In certain embodiment, the particleis further conjugated to a targeting moiety and/or siRNA.

In certain embodiments, the disclosure relates to methods comprisingadministering a pharmaceutical composition comprising a particleconjugate disclosed herein to a subject in need thereof. In certainembodiments, the subject is diagnosed with cancer, a viral infection, ora bacterial infection.

In certain embodiments, the disclosure relates to the production of amedicament or pharmaceutical composition comprising particles disclosedherein and a pharmaceutically acceptable excipient for use in treatingcancer or a viral or bacterial infection. Typically, the pharmaceuticalcomposition is in the form of a buffered saline solution, capsule, pill,or tablet.

In certain embodiments, conjugated particles are used in detecting metalions and other small molecule analytes. In certain embodiments, thedisclosure relates to methods of mixing particles disclosed herein and asample containing metal ions or other molecules that bind nucleic acidson the particles and measuring binding.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the disclosure, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A schematically illustrates DzNP synthesis and catalysis strategyused to generate DNAzyme-AuNP conjugates (DzNPs). A binary mixture oftwo oligonucleotides, DNAzymes and passivating strands, were used tocontrol the steric environment at the particle surface. The catalyticactivity of DzNPs was deter-mined by measuring the rate of fluorescenceincrease, which represents the rate of substrate hydrolysis andde-quenching. The cleaving activity of DzNPs was determined by measuringthe rate of fluorescence increase, which represents the rate ofsubstrate hydrolysis and de-quenching. 10-23 DNAzyme is AGCAACATCGATCGG(SEQ ID NO:1) which is within TCTCTCAGCAACATCGATCGGACCCACG (SEQ ID NO:2) which hybridizes and cleaves CGTGGGrUrAGAGAGAG (SEQ ID NO: 3).

FIG. 1B is data. Top is a representative kinetic plot showing the rateof catalysis for 4.2 nM DzT10NP (closed circles) and DzT10 (triangles)en-zymes. Middle is a plot showing the DNAzyme surface density for T10passivated DzNPs as a function of the mole fraction of DNAzyme addedduring the NP functionalization. Bottom is a plot showing kobs for thehydrolysis of a series of DzNPs that vary in DNAzyme surface density at[Mg²⁺]=10 mM. The horizontal red line denotes the activity of free DzT₁₀(4.2 nM). The diagonal dashed line is a best fit (R₂=0.99) through thelower density DzNP data points (≦60 DNAzymes/particle). All error barsrepresent the standard deviation of three measurements.

FIG. 2 illustrates and shows data on DNAzyme release from goldnanoparticles. (A) Schematic showing DTT displacement (left) or laserirradiation (right) to release Dz_(REV)T₁₀ from the particle to enhancecatalysis. (B) A kinetic plot showing rate of catalysis of Dz_(REV)T₁₀NP(open circles) and DTT displaced Dz_(REV)T₁₀ (open triangles) from theparticle. (C) A plot showing k_(obs) for the hydrolysis normalized toDz_(REV)T₁₀NP for a series of particles irradiated at 532 nm fordifferent times (intact particle=open circle and partially releasedDz=open triangles: 10 Hz, 4 ns, 100 mW

FIG. 3 shows data on gene regulation using DzNP. Top is a bar graphshowing the relative catalytic activity of DzT₁₀ and DzT₁₀NP afterexposure to DNase I for 120 min at 37° C. Bottom is a real-time PCRanalysis of GDF15 mRNA expression of HCC1954 cells grown in a 12-wellplate, which were treated with catalytic (DzGDFNP) and non-catalytic(i-DzGDFNP) DzNPs targeting GDF15 mRNA and non-specific catalytic(DzNCNP) DzNPs at a NP concentration of 5 nM for 48 hrs.

FIG. 4 is a plot showing the DNAzyme surface density of DzNPs asdetermined by two independent techniques. The first method is based onan oligonucleotide fluorescence quantification kit (open circles). Thesecond approach measures the catalytic activity of DTT-released DNAzymeand quantifies the concentration using a standard calibration curve (redcircles). Error bars represents the standard deviation of threemeasurements.

FIG. 5 shows data for DNAzyme-nanoparticle mediated knock down of GDF15.HCC1954 cells were treated with nanoparticles conjugated to DNAzyme forGDF15 at a concentration of 5 nM for 48 hrs. Total RNA was isolated andexpression of GDF15 was determined by real-time PCR analysis. Valueswere normalized to housekeeping gene RPLPO. Fold expression relative tonon-targeting control (NC) is shown. Error bars represent standarddeviation between triplicates.

FIG. 6 shows data on the activity of DzNP synthesized using a variety ofdifferent linkers. The k_(obs) values were normalized to the activity ofDz((EG₆)PO₃)₃NPs ([MgCl₂]=10 mM). Error bars are the standard deviationof three measurements.

FIG. 7 shows data on the catalytic activity of DzNP synthesized using avariety of different linkers. The k_(obs) values were normalized to theactivity of Dz((EG₆)PO₃)₃NPs ([MgCl₂]=10 mM). Error bars are thestandard deviation of three measurements.

FIG. 8 shows a bar graph showing the normalized catalytic activity ofDz_(GFP)T₁₀NP (4.2 nM) and Dz_(GFP)T₁₀ (4.2 nM) normalized toDz_(GFP)T₁₀ at 25° C.

DETAILED DESCRIPTION

This disclosure relates to particles conjugated to therapeutic nucleicacids. In certain embodiments, the nucleic acid comprises a sequencethat catalytically cleaves RNA, e.g., DNAzyme or RNAzyme. In certainembodiments, the particles contain nucleic acids with both DNAzymeand/or RNAzyme and siRNA sequences. The cleaving nucleic acidsoptionally comprise a sequence functioning to hybridize to a target ofinterest and/or the particles are further conjugated to a targetingmoiety. In certain embodiments, conjugated particles are used in thetreatment or prevention of cancer or viral infections or bacterialinfections. In certain embodiments, conjugated particles are used indetecting metal ions and other small molecule analytes.

I. Particles Conjugated to Cleaving Nucleic Acids

In certain embodiments, the disclosure relates to particles conjugatedto a cleaving nucleic acid wherein the nucleic acid comprises a sequencethat cleaves RNA. Single-stranded nucleic acids can fold into tertiarystructures and act as catalysis similar to enzymes made of protein.Ribozymes, RNAzymes, and deoxyribozymes, DNAzymes, have been isolatedfrom naturally occurring molecules and optimized from random-sequencepopulations using in vitro selection. A combinatorial strategy may beused to create numerous classes of nucleic acid-cleaving DNAzymes andRNAzymes. DNAzymes and RNAzymes often, but not exclusively, catalyzecleavage of the RNA 3′,5′-phosphodiester linkage by promoting aninternal transesterification reaction to produce 2′,3′-cyclic phosphateand 5′-hydroxyl termini.

In certain embodiments, the disclosure relates to particles conjugatedto a cleaving nucleic acid such as DNAzyme 10-23. The DNAzyme 10-23 iscomprised of a sequence of DNA that will cleave mRNA strands thatcontain an unpaired purine-pyrimidine pair. The DNAzyme 10-23 istypically flanked by a recognition sequence that will specificallyrecognize a short region of the target mRNA. Therefore, the DNAzyme willrecognize the complementary mRNA, hybridize, cleave at a site, andconsequently alter the expression of protein targets.

In certain embodiments, this disclosure contemplates that the cleavingnucleic acids comprise sequences of DNAzymes 8-17 and 10-23. Santoro &Joyce disclosed a general purpose RNA-cleaving DNAzymes 8-17 and 10-23.See PNSA, 1997, 94 (9), 4262-4266, hereby incorporated by reference.

In certain embodiments, this disclosure contemplates that the cleavingnucleic acids comprise 8-imidazolyl modified deoxy adenosines RNaseAmimicking DNAzymes. Perrin et al., disclose modified DNAzymes 20-49containing amine, guanidine, and imidazole-modified dNTPs. Org BiomolChem 2011, 9 (7), 2266-2273, hereby incorporated by reference.

In certain embodiments, this disclosure contemplates that the cleavingnucleic acids is DNAzyme pH5DZ1. Li et al., disclose DNAzymes pH5DZ1.See Biochemistry, 2009, 48 (31):7383-7391, hereby incorporated byreference. Li et al, also disclose DNAzyme classes with large catalyticdomains (>40 nucleotides) utilizing three-way or four-way junctionstructural frameworks. See Mol Biosyst 2011, 7 (7), 2139-2146, herebyincorporated by reference.

In certain embodiments, the cleaving nucleic acid is 10MD9, 10MD1,10MD14, and 10MD5. Chandra et al., disclose 10MD9, 10MD1, 10MD14, and10MD5. See Nat Chem Biol 2009, 5 (10), 718-720, hereby incorporated byreference.

In certain embodiments, the cleaving nucleic acid is a bipartiteDNAzyme. Feldman and Sen disclose bipartite DNAzymes suitable for thesequence-specific cleavage of RNA. See J Mol Biol 2001, 313 (2): 283-294and Chembiochem, 2006, 7 (1): 98-105, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid is a DNAzyme of the Nafamily. A DNAzyme of the Na family generally contains a consensussequence ACCCAAGAAGGGGTG (SEQ ID NO:4). In certain embodiments, thecleaving nucleic acid comprises SEQ ID NO:4 and GCX¹TX²ACX³X⁴X⁵AT (SEQID NO:5) wherein X is any amino acid. Geyer & Sen disclose DNAzymes ofthe Na family. See Chem Biol, 1997, 4 (8): 579-593, hereby incorporatedby reference.

In certain embodiments, the cleaving nucleic acid is a DNAzyme HD2.Breaker & Roth disclose HD2. See. PNAS, 1998, 95 (11): 6027-6031, herebyincorporated by reference

In certain embodiments, the cleaving nucleic acid is a DNAzyme pH3Dz1.Li et al., disclose pH3DZ1. See Can J Chem 2007, 85 (4), 261-273, herebyincorporated by reference.

In certain embodiments, the cleaving nucleic acid is a DNAzymecomprising AATTCCGTAGGTCCAGTG (SEQ ID NO:6) and ATCTCCTCCTGTTC (SEQ IDNO:7. Adriaens and Vannela disclosed RNA-cleaving DNAzymes containingSEQ ID NO: 6 and SEQ ID NO:7. See Environ Eng Sci 2007, 24 (1), 73-84,hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid is DNAzyme MG5.Peracchi disclosed DNAzyme MG5 deoxyribozyme. See J Biol Chem 2000, 275(16), 11693-11697, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid is DNAzyme 17E. Incertain embodiments, the cleaving nucleic acid comprises X₃AGCY₃TCGAA(SEQ ID NO: 8), or TX₃AGCY₃TCGAAATAGT (SEQ ID NO: 9) wherein X₃ and Y₃are complimentary. Li et al., disclose DNAzyme 17E and other variants.See Nucleic Acids Res, 2000, 28 (2), 481-488, hereby incorporated byreference.

In certain embodiments, the cleaving nucleic acid is DNAzyme 614.Carrigan et al., disclosed DNAzyme 614. See Biochemistry, 2004, 43 (36),11446-11459, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to mRNA of EGR-1. Fahmy et al., disclose DNAzymestargeting early growth response (EGR-1) mRNA. See Nat Med 2003, 9 (8),1026-1032 and Santiago et al., Nat Med, 1999, 5 (12), 1438-1438, bothhereby incorporated by reference. In certain embodiments, the cleavingnucleic acid comprises a segment that hybridizes to human EGR-1 mRNA.Mitchell et al., disclose a DNAzyme targeting EGR-1. See Nucleic AcidsRes 2004, 32 (10), 3065-3069, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to c-Jun mRNA, e.g., Dz13. Zhang et al., disclosedeoxyribozymes targeting c-Jun. See J Biol Chem 2002, 277 (25),22985-22991, J Natl Cancer 12004, 96 (9), 683-696, and Oncogene 2006, 25(55), 7260-7266, all hereby incorporated by reference. Fahmy et al.,disclose DNAzyme Dz13 that cleaves human c-Jun mRNA. See Nat Biotechnol2006, 24 (7), 856-863, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acids comprise a segmentthat hybridizes to LMP1 mRNA. Lu et al., disclose EBV LMP1 targetedDNAzymes. See Cancer Gene Ther 2005, 12 (7), 647-654, herebyincorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to hepatitis B virus X gene open reading frame. Hou etal., disclose inhibition of hepatitis B virus X gene expression by aDNAzymes 10-23. See Antivir Res 2006, 72 (3), 190-196, herebyincorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to RNA sequences encoding the HCV core protein Trepanieret al., disclose cleavage of intracellular hepatitis C RNA in the viruscore protein coding region by deoxyribozymes. See J Viral Hepatitis2006, 13 (2), 131-138, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to HIV RNA sequences, e.g., encoding HIV-1 gag, nef,rev, env or tat. Sriram & Banerjea disclose DNAzymes interference withHIV-1-specific gene expression. See Biochem J 2000, 352, 667-673, herebyincorporated by reference. See also Dash & Banerjea, Oligonucleotides2004, 14 (1), 41-47, Sood et al., Oligonucleotides 2007, 17 (1),113-121, and Unwalla et al., Antivir Res 2006, 72 (2), 134-144, Sood etal., Aids 2007, 21 (1), 31-40, and Jakobsen et al., Retrovirology 2007,4, all hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to mRNA of human telomere reverse transcriptase. Yuan etal., disclose DNAzymes targeting the telomerase mRNA. See Int J BiochemCell B 2007, 39 (6), 1119-1129, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to mRNA of influenza virus, e.g., PB2 mRNA of influenzaA. Takahashi et al., disclose DNAzymes that targets influenza virus AmRNA. See Febs Lett 2004, 560 (1-3), 69-74, hereby incorporated byreference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to mRNA mycobacterium tuberculosis, e.g., mRNA ofIsocitrate lyase (ICL). Li et al., disclose DNAzymes targeting the iclgene. See Oligonucleotides 2005, 15 (3), 215-222, hereby incorporated byreference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to bacterial mRNA, e.g., beta-lactamase mRNA. Chen etal., disclose DNAzymes targeted to beta-lactamase mRNA. SeeOligonucleotides 2004, 14 (2), 80-89, hereby incorporated by reference.See also Hou et al., Acta Pharmacol Sin 2007, 28 (11), 1775-1782, herebyincorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to human platelet type 12 lipoxygenase mRNA. Liu et al.,disclose cleaving DNAzyme to mRNA of platelet-type 12-lipoxygenase. SeeBiochem Bioph Res Co 2001, 284 (4), 1077-1082, hereby incorporated byreference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to the translation initiation region of c-myc RNA. Sunet al., disclose a DNAzyme targeting c-myc RNA. See J Biol Chem 1999,274 (24), 17236-17241, hereby incorporated by reference. See also, Punet al., Cancer Biol Ther 2004, 3 (7), 641-650, hereby incorporated byreference.

In certain embodiments, the cleaving nucleic acid comprises a 3′ endmodified such that last two nucleotides are connected by aphosphodiester linkage between the 3′ positions of each nucleotides. SeeDass et al., Nucleic A 2002, 12 (5), 289-299, hereby incorporated byreference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to ornithine decarboxylase (ODC). Ackermann et al.,disclose DNAzyme to ornithine decarboxylase mRNA. See Biochemistry,2005, 44 (6), 2143-2152, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to MecR1 mRNA, e.g., PS-DRz147. Hou et al., disclosedPS-DRz147. See Acta Pharmacol Sin 2006, 27, 59-59, hereby incorporatedby reference

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to genomic RNA sequence of the RSV nucleocapsid protein,e.g., DZ1133. Zhou et al., disclose DNAzyme DZ1133. See Virus Res 2007,130 (1-2), 241-248, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to the loop region of the 5′UTR of SARS-CoV, e.g.,Dv-104. Wu et al., disclose that DNAzyme, DZ-104 can specifically targetthe 5′-untranslated region of severe acute respiratory syndromeassociated coronavirus (SARS-CoV). See J Gene Med 2007, 9 (12),1080-1086, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to survivin mRNA. Liang et al., disclose DNAzymecleavage of survivin mRNA. See J Gastroen Hepatol 2005, 20 (10),1595-1602, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to TGF-beta mRNA. See Isaka et al., disclose DNAzyme forTGF-beta. See Kidney Int, 2004, 66 (2), 586-590, hereby incorporated byreference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to Twist mRNA. Hjiantoniou et al., discloseDNazyme-mediated cleavage of Twist transcripts. See Biochem Bioph Res Co2003, 300 (1), 178-181, hereby incorporated by reference.

In certain embodiments, the cleaving nucleic acid comprises a segmentthat hybridizes to mRNA of VEGFR2. Zhang et al., discloses a DNAzymethat targets vascular endothelial growth factor receptor 2. See CancerRes 2002, 62 (19), 5463-5469, hereby incorporated by reference.

II. Therapeutic Applications and Combination Therapies

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising particle conjugates disclosed herein foradministration to a subject for the prevention or treatment of acondition or disease. In certain embodiments, the disclosure relates tomethods of treating or preventing cancer or a viral infection or abacterial infection comprising administering an effective amount of apharmaceutical composition disclosed herein to a subject.

In certain embodiments, the subject may be diagnosed with a cancer, atrisk of cancer, diagnosed with a tumor. In certain embodiments, thecancer is selected from brain, lung, cervical, ovarian, colon, breast,gastric, skin, ovarian, pancreatic, prostate, neck, and renal cancer.

In some embodiments, the pharmaceutical composition is administered incombination with a second anticancer agent, and the second anticanceragent may be selected from temozolamide, bevacizumab, procarbazine,lomustine, vincristine, gefitinib, erlotinib, docetaxel, cis-platin,5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate,cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin,daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin andmithramycin, vinblastine, vindesine, vinorelbine, taxol, taxotere,etoposide, teniposide, amsacrine, topotecan, camptothecin, bortezomib,anagrelide, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene,fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone,goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole,vorazole, exemestane, finasteride, marimastat, trastuzumab, cetuximab,dasatinib, imatinib, combretastatin, thalidomide, and/or lenalidomide orcombinations thereof.

In certain embodiment, the pharmaceutical composition is administered incombination with an siRNA.

In certain embodiment, the subject may be pre- or post-operativelyprovided radiation therapy. In certain embodiments, contemplated methodscomprise administering the particle conjugated and utilizingphototherapy, e.g., releasing the attached nucleic acid in a target areaof the by exposing the subject to electromagnetic radiation underconditions such that the administered particle releases nucleic acidsconjugated to the particle.

In certain embodiments, the subject may be diagnosed with a viral orbacterial infection, at risk of viral or bacterial infection, orexhibiting symptoms of a viral or bacterial infection. In certainembodiments, the subject is diagnosed with an RNA based viral infection,e.g., an RNA virus, or utilizes an RNA intermediated.

In some embodiments, the subject is diagnosed with influenza A virusincluding subtype H1N1, influenza B virus, influenza C virus, rotavirusA, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SARS coronavirus,human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) Types16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, parvovirus B19,molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cellpolyomavirus, coxsackie A virus, norovirus, Rubella virus, lymphocyticchoriomeningitis virus (LCMV), yellow fever virus, measles virus, mumpsvirus, respiratory syncytial virus, rinderpest virus, Californiaencephalitis virus, hantavirus, rabies virus, ebola virus, marburgvirus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2),varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus(CMV), herpes lymphotropic virus, roseolovirus, Kaposi'ssarcoma-associated herpesvirus, hepatitis A (HAV), hepatitis B (HBV),hepatitis C (HCV), hepatitis D (HDV), hepatitis E (HEV), humanimmunodeficiency virus (HIV), The Human T-lymphotropic virus Type I(HTLV-1), Friend spleen focus-forming virus (SFFV) or XenotropicMuLV-Related Virus (XMRV).

In some embodiments, the subject is diagnosed with gastroenteritis,acute respiratory disease, severe acute respiratory syndrome, post-viralfatigue syndrome, viral hemorrhagic fevers, acquired immunodeficiencysyndrome, or hepatitis.

In some embodiments, the disclosure relates to treating a viralinfection by administering a composition comprising particles disclosedherein in combination with a second antiviral agent, e.g., abacavir,acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen,arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir,darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz,emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen,fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, immunovir,idoxuridine, imiquimod, indinavir, inosine, interferon type III,interferon type II, interferon type I, lamivudine, lopinavir, loviride,maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir,oseltamivir (Tamiflu), peginterferon alfa-2a, penciclovir, peramivir,pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine,ritonavir, pyramidine, saquinavir, stavudine, tenofovir, tenofovirdisoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada,valaciclovir (Valtrex), valganciclovir, vicriviroc, vidarabine,viramidine zalcitabine, zanamivir (Relenza), and/or zidovudine. Incertain embodiments, the subject is administered a pharmaceuticalcomposition comprising particles disclosed herein and a second antiviralagent.

In certain embodiments, the subject is treated for a bacterial infectionin combination with another antibiotic such as sulphadiazine,sulfones—[dapsone (DDS) and paraminosalicyclic (PAS)], sulfanilamide,sulfamethizole, sulfamethoxazole, sulfapyridine, trimethoprim,pyrimethamine, nalidixic acids, norfloxacin, ciproflaxin, cinoxacin,enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin,lomefloxacin, moxifloxacin, ofloxacin, pefloxacin, sparfloxacin,trovafloxacin, penicillins (amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, hetacillin,oxacillin, mezlocillin, penicillin G, penicillin V, piperacillin),cephalosporins (cefacetrile, cefadroxil, cefalexin, cefaloglycin,cefalonium, cefaloridin, cefalotin, cefapirin, cefatrizine, cefazaflur,cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor,cefonicid, ceforanide, Cefprozil, cefuroxime, cefuzonam, cefmetazole,cefoteta, cefoxitin, cefcapene, cefdaloxime, cefdinir, cefditoren,cefetamet, cefixime, cefmenoxime, cefodizime, cefoperazone, cefotaxime,cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefteram, ceftibuten,ceftiofur, ceftiolen, ceftizoxime, ceftriaxone, cefoperazone,ceftazidime, cefepime), moxolactam, carbapenems (imipenem, ertapenem,meropenem), monobactams (aztreonam), oxytetracycline, chlortetracycline,clomocycline, demeclocycline, tetracycline, doxycycline, lymecycline,meclocycline, methacycline, minocycline, rolitetracycline,chloramphenicol, amikacin, gentamicin, framycetin, kanamycin, neomicin,neomycin, netilmicin, streptomycin, tobramycin, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,telithromycin, polymyxin-B, colistin, bacitracin, tyrothricinnotrifurantoin, furazolidone, metronidazole, tinidazole, isoniazid,pyrazinamide, ethionamide, nystatin, amphotericin-B, hamycin,miconazole, clotrimazole, ketoconazole, fluconazole, rifampacin,lincomycin, clindamycin, spectinomycin, chloramphenicol, clindamycin,colistin, fosfomycin, loracarbef, metronidazole, nitrofurantoin,polymyxin B, polymyxin B sulfate, procain, spectinomycin, tinidazole,trimethoprim, ramoplanin, teicoplanin, vancomycin, trimethoprim,sulfamethoxazole, and/or nitrofurantoin.

III. Metal Particles, Coatings, and Preparation

This disclosure relates to particles conjugated to a cleaving nucleicacid wherein the nucleic acid comprises a sequence that cleaves RNA,e.g., DNAzyme or RNAzyme. In certain embodiments, the particle comprisesor consists essentially of a metal such as gold, silver, iron, or ironoxide. Typically, the particle is a metal nanoparticle. In someembodiments, the cleaving nucleic acid is conjugated to the metalparticle through surface coated polymer.

Contemplated particles include pegylated colloidal gold and iron oxidenanoparticles. See Qian et al., Nature Biotechnology, 2008, 26, 83-90,Hadjipanayis et al., Cancer Research, 2010, 70(15):6303-6312, and Penget al., Int J Nanomedicine. 2008 September; 3(3): 311-321, all herebyincorporated by reference. A couple of approaches may be used for thechemical synthesis of contemplated gold nanoparticles. Alkanethiols maybe used to stabilize gold particles. See, e.g., Brust et al., J ChemSoc, Chem Commun, 1994, 801-02 and Templeton et al., Acc Chem Res, 2000,33, 27, all hereby incorporated by reference. In another approach, oneuses sodium citrate as a reducing agent and stabilizing ligand. SeeTurkevich et al., Discuss Faraday Soc, 1951, 11, 55, hereby incorporatedby reference. The particle size can be controlled by the goldprecursor/citrate molar ratio. Kairdolf & Nie disclose the production ofmultidentate-protected colloidal gold nanoparticles. See J. Am. Chem.Soc. 2011, 133, 7268-7271, hereby incorporated by reference.

Nanoparticles are typically prepared with a mean particle diameter of4-100 nm. Iron-oxide nanoparticles (IONPs) may be prepared by aging astoichiometric mixture of ferrous and ferric salts in aqueous mediaunder basic conditions. Control over particle size (2-20 nm) and shapeis provided by adjusting the pH, ionic strength and the concentration ofthe growth solution. The nanoparticles can be functionalized in situusing additives such as organic compounds (e.g. sodium citric) orpolymers (e.g. dextran, polyvinyl alcohol). Other metals such as gold,cobalt, nickel, and manganese may be incorporated into the material.

High-temperature decomposition of Fe(CO)₅ in organic solvents is anotherway to prepare IONPs. Size (3-19 nm) can be varied using alternativetemperatures. Flame spray pyrolysis yields a range of magnetite,maghemite and wustite (FeO) particles IONPs. Iron precursor such asFe(CO)₅ and Fe(NO₃)₃ may be used. Flame spray pyrolysis can be used toproduce different nanoparticles (TiO₂, ZrO₂, silica, etc.) as well ashybrid particles (e.g. silica-IONPs).

Hydroxyl groups on the IONP provide a place for synthetic attachment ofdifferent functional groups. A range of chemistries can be used tostabilize metal nanoparticles, exploiting electrostatic, hydrophobic,chelating and covalent interactions. Carboxylic acid groups can interactwith the surface of IONPs by coordination processes. IONP synthesis inorganic solvents is typically conducted in oleic acid. A polymer coatingon the IONPs is preferred. Polymer attachment to the IONP surface by aninitiator fixed to the surface of the IONPs and the polymer is grownfrom the surface. Alternatively, a functional, pre-formed polymer isgrafted onto IONPs in situ. Copolymers with hydrophobic groups,carboxylic acid groups, polyethylene glycols, or amine groups arecontemplated

Conjugating cleaving nucleic acids to the polymers can be accomplishedusing a variety of methods. For example, a primary amine containingnucleic acid may be conjugated to the carboxylic acid groups on a coatedpolymer mediated by a coupling reagent such as EDAC. See, e.g., Yang etal., Small, 2009, 5(2):235-43, hereby incorporated by reference. Othercoupling methods are contemplated, e.g., the avidin/streptavidin-biotininteractions may be used, e.g., streptavidin may be coupled to thecoated polymer surface and biotin may be linked to the cleaving nucleicacid.

IV. Pharmaceutical Compositions

Generally, for pharmaceutical use, the compositions with the particleconjugates may be formulated as a pharmaceutical preparation comprisingparticle conjugates and a pharmaceutically acceptable carrier, diluentor excipient and/or adjuvant, and optionally one or more furtherpharmaceutically active compositions.

The pharmaceutical preparations of the disclosure are preferably in aunit dosage form, and may be suitably packaged, for example in a box,blister, vial, bottle, sachet, ampoule or in any other suitablesingle-dose or multi-dose holder or container (which may be properlylabeled); optionally with one or more leaflets containing productinformation and/or instructions for use. Generally, such unit dosageswill contain between 1 and 1000 mg, and usually between 5 and 500 mg,e.g. about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

The compositions can be administered by a variety of routes includingthe oral, ocular, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal routes, depending mainly on the specificpreparation used. In certain embodiments, the disclosure contemplatesintravenously-delivery of an aqueous saline buffer.

The embodiments will generally be administered in an “effective amount”,by which is meant any amount of a composition that, upon suitableadministration, is sufficient to achieve the desired therapeutic orprophylactic effect in the subject to which it is administered. Usually,depending on the condition to be prevented or treated and the route ofadministration, such an effective amount will usually be between 0.01 to1000 mg per kilogram body weight of the patient per day, more oftenbetween 0.1 and 500 mg, such as between 1 and 250 mg, for example about5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight of thepatient per day, which may be administered as a single daily dose,divided over one or more daily doses. The amount(s) to be administered,the route of administration and the further treatment regimen may bedetermined by the treating clinician, depending on factors such as theage, gender and general condition of the patient and the nature andseverity of the disease/symptoms to be treated. Reference is again madeto U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No.6,369,087 and U.S. Pat. No. 6,372,733 and the further referencesmentioned above, as well as to the standard handbooks, such as thelatest edition of Remington's Pharmaceutical Sciences.

For an oral administration form, the composition can be mixed withsuitable additives, such as excipients, stabilizers or inert diluents,and brought by means of the customary methods into the suitableadministration forms, such as tablets, coated tablets, hard capsules,aqueous, alcoholic, or oily solutions. Examples of suitable inertcarriers are gum arabic, magnesia, magnesium carbonate, potassiumphosphate, lactose, glucose, or starch, in particular, corn starch. Inthis case, the preparation can be carried out both as dry and as moistgranules. Suitable oily excipients or solvents are vegetable or animaloils, such as sunflower oil or cod liver oil. Suitable solvents foraqueous or alcoholic solutions are water, ethanol, sugar solutions, ormixtures thereof. Polyethylene glycols and polypropylene glycols arealso useful as further auxiliaries for other administration forms. Asimmediate release tablets, these compositions may containmicrocrystalline cellulose, dicalcium phosphate, starch, magnesiumstearate and lactose and/or other excipients, binders, extenders,disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, the compositions maybe prepared according to techniques well-known in the art ofpharmaceutical formulation and may be prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art. Suitablepharmaceutical formulations for administration in the form of aerosolsor sprays are, for example, solutions, suspensions or emulsions of thecompositions of the disclosure or their physiologically tolerable saltsin a pharmaceutically acceptable solvent, such as ethanol or water, or amixture of such solvents. If required, the formulation can alsoadditionally contain other pharmaceutical auxiliaries such assurfactants, emulsifiers and stabilizers as well as a propellant.

For subcutaneous or intravenous administration, the compositions, ifdesired with the substances customary therefore such as solubilizers,emulsifiers or further auxiliaries are brought into solution,suspension, or emulsion. The compositions can also be lyophilized andthe lyophilizates obtained used, for example, for the production ofinjection or infusion preparations. Suitable solvents are, for example,water, physiological saline solution or alcohols, e.g. ethanol,propanol, glycerol, sugar solutions such as glucose or mannitolsolutions, or mixtures of the various solvents mentioned. The injectablesolutions or suspensions may be formulated according to known art, usingsuitable non-toxic, parenterally-acceptable diluents or solvents, suchas mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodiumchloride solution, or suitable dispersing or wetting and suspendingagents, such as sterile, bland, fixed oils, including synthetic mono- ordiglycerides, and fatty acids, including oleic acid.

In certain embodiments, it is contemplated that these compositions canbe extended release formulations. Typical extended release formationsutilize an enteric coating. Typically, a barrier is applied to oralmedication that controls the location in the digestive system where itis absorbed. Enteric coatings prevent release of medication before itreaches the small intestine. Enteric coatings may contain polymers ofpolysaccharides, such as maltodextrin, xanthan, scleroglucan dextran,starch, alginates, pullulan, hyaloronic acid, chitin, chitosan and thelike; other natural polymers, such as proteins (albumin, gelatin etc.),poly-L-lysine; sodium poly(acrylic acid);poly(hydroxyalkylmethacrylates) (for example poly(hydroxyethylmethacrylate)); carboxypolymethylene (for example Carbopol™); carbomer;polyvinyl pyrrolidone; gums, such as guar gum, gum arabic, gum karaya,gum ghatti, locust bean gum, tamarind gum, gellan gum, gum tragacanth,agar, pectin, gluten and the like; poly(vinyl alcohol); ethylene vinylalcohol; polyethylene glycol (PEG); and cellulose ethers, such ashydroxy methylcellulose (HMC), hydroxyethylcellulose (HEC),hydroxypropylcellulose (HPC), methylcellulose (MC), ethylcellulose (EC),carboxyethylcellulose (CEC), ethylhydroxy ethylcellulose (EHEC),carboxymethylhydroxyethylcellulose (CMHEC),hydroxypropylmethyl-cellulose (HPMC), hydroxypropylethylcellulose (HPEC)and sodium carboxymethylcellulose (Na CMC); as well as copolymers and/or(simple) mixtures of any of the above polymers. Certain of theabove-mentioned polymers may further be crosslinked by way of standardtechniques.

The choice of polymer will be determined by the nature of the activeingredient/drug that is employed in the composition of the disclosure aswell as the desired rate of release. In particular, it will beappreciated by the skilled person, for example in the case of HPMC, thata higher molecular weight will, in general, provide a slower rate ofrelease of drug from the composition. Furthermore, in the case of HPMC,different degrees of substitution of methoxy groups and hydroxypropoxylgroups will give rise to changes in the rate of release of drug from thecomposition. In this respect, and as stated above, it may be desirableto provide compositions of the disclosure in the form of coatings inwhich the polymer carrier is provided by way of a blend of two or morepolymers of, for example, different molecular weights in order toproduce a particular required or desired release profile.

Microspheres of polylactide, polyglycolide, and their copolymerspoly(lactide-co-glycolide) may be used to form sustained-releasedelivery systems. Particle conjugates can be entrapped in thepoly(lactide-co-glycolide) microsphere depot by a number of methods,including formation of a water-in-oil emulsion with water and organicsolvent (emulsion method), formation of a solid-in-oil suspension withparticle dispersed in a solvent-based polymer solution (suspensionmethod), or by dissolving the particle in a solvent-based polymersolution (dissolution method). One can attach poly(ethylene glycol) toparticles (pegylation) to increase the in vivo half-life.

V. Targeting Moieties

Within certain embodiments, particles conjugated to cleaving nucleicacids further comprising a targeting moiety in order to target theparticle to a physiological tissue or group of cells. Typically,diseased cells overexpress a specific cell surface marker, e.g., HER 2for breast cancer cells. Antibodies or other molecules with bindingaffinity to these markers may be conjugated to the particles in order torestrict the movement of the particle to the location of the cells afteradministration.

In certain embodiments, the targeting moiety is a monoclonal antibody toHER-2, e.g., Herceptin, that targets HER-2 receptors for use in treatingbreast cancer. See Lee et al., Nat Med, 2007, 13:95-9; Artemov et al.,Magn Reson Med, 2003, 49:403-8; and Huh et al., J Am Chem Soc, 2005,127:12387-91, all hereby incorporated by reference in their entirety.

In certain embodiments, the targeting moiety is a monoclonalantibody-610 that targets a surface antigen for use in treating coloncarcinoma. See Cerdan et al., Magn Reson Med, 1989, 12:151-63 1989,hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is an antibody tocarcinoembryonic antigen (CEA) that targets CEA for use in treatingcolon tumors. See Tiefenauer et al., Magn Reson Imaging, 1996,14:391-402, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is a monoclonal antibody L6that targets a surface antigen for use in treating intracranial tumor.See Remsen et al., Am J Neuroradiol, 1996, 17:411-18, herebyincorporated by reference in its entirety.

In certain embodiments, the targeting moiety is transferrin that targetstransferrin receptor for use in treating carcinoma. See Kresse et al.,Magn Reson Med, 1998, 40:236-42, hereby incorporated by reference in itsentirety.

In certain embodiments, the targeting moiety is the EPPT peptide thattargets underglycosylated mucin-1 antigen (uMUC-1) for use in treatingbreast, colon, pancreas and lung cancer. See Moore et al., Cancer Res,2004, 64:1821-7, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is folic acid that targetsfolate receptor for use in treating mouth carcinoma and cervical cancer.See Chen et al., PDA J Pharm Sci Technol, 2007, 61:303-13; Sun et al.,Small, 2006, 4:372-9; and Sonvico et al., Bioconjug Chem, 2005,16:1181-8, all hereby incorporated by reference in their entirety.

In certain embodiments, the targeting moiety is methotrexate thattargets folate receptor for use in treating cervical cancer. See Kohleret al., Langmuir, 2005, 21:8858-64, hereby incorporated by reference inits entirety.

In certain embodiments, the targeting moiety is a monoclonal antibody A7that targets colorectal tumor antigen for use in treating colorectalcarcinoma. See Toma et al., Br J Cancer, 2005, 93:131-6, herebyincorporated by reference in its entirety.

In certain embodiments, the targeting moiety is chlorotoxin peptide thattargets membrane-bound matrixmetalloproteinase-2 (MMP-2) for use intreating glioma. See Veiseh et al., Nano Lett, 2005, 5:1003-8, herebyincorporated by reference in its entirety.

In certain embodiments, the targeting moiety is F3 peptide that targetssurface-localized tumor vasculature for use in treating glioma. SeeReddy et al., Clin Cancer Res, 2006, 12:6677-86, hereby incorporated byreference in its entirety.

In certain embodiments, the targeting moiety is RGD or RGD4C thattargets integrins for use in treating melanoma and epidermoid carcinoma.See Zhang et al., Cancer Res, 2007, 67:1555-62 and Uchida et al., J AmChem Soc, 2006, 128:16626-33, both hereby incorporated by reference intheir entirety.

In certain embodiments, the targeting moiety is luteinizing hormonereleasing hormone (LHRH) that targets LHRH receptor for use in treatingbreast cancer. See Leuschner et al., Breast Cancer Res Treat, 2006,99:163-76, hereby incorporated by reference in its entirety.

In certain embodiments, the targeting moiety is CREKA peptide thattargets clotted plasma proteins for use in treating breast cancer. SeeSimberg et al., Proc Natl Acad Sci USA, 2007, 104:932-6, herebyincorporated by reference in its entirety.

In certain embodiments, the targeting moiety is an antibody to prostatespecific membrane antigen (PSMA) that targets PSMA for use in treatingprostate cancer. See Serda et al., Mol Imaging, 2007, 6:277-88, herebyincorporated by reference in its entirety.

In certain embodiments, the disclosure contemplates targeting moietiesor proteins in any of the disclosed embodiments that are antibodies orfragments or chimera, antibody mimetics, or aptamers or any molecularentity that selectively binds targets that are more prevalent on cancercells.

Numerous methods known to those skilled in the art are available forobtaining antibodies or antigen-binding fragments thereof. For example,antibodies can be produced using recombinant DNA methods (U.S. Pat. No.4,816,567). Monoclonal antibodies may also be produced by generation ofhybridomas in accordance with known methods. Hybridomas formed in thismanner are then screened using standard methods, such as enzyme-linkedimmunosorbent assay (ELISA) and surface plasmon resonance analysis, toidentify one or more hybridomas that produce an antibody thatspecifically binds with a specified antigen. Any form of the specifiedantigen may be used as the immunogen, e.g., recombinant antigen,naturally occurring forms, any variants or fragments thereof, as well asantigenic peptide thereof.

The modular structure of antibodies makes it possible to remove constantdomains in order to reduce size and still retain antigen bindingspecificity. Engineered antibody fragments allow one to create antibodylibraries. A single-chain antibody (scFv) is an antibody fragment wherethe variable domains of the heavy (V_(H)) and light chains (V_(L)) arecombined with a flexible polypeptide linker. The scFv and Fab fragmentsare both monovalent binders but they can be engineered into multivalentbinders to gain avidity effects. One exemplary method of makingantibodies and fragments includes screening protein expressionlibraries, e.g., phage or ribosome display libraries. Phage display isdescribed, for example, in U.S. Pat. No. 5,223,409.

In addition to the use of display libraries, the specified antigen canbe used to immunize a non-human animal, e.g., a rodent, e.g., a mouse,hamster, or rat. In one embodiment, the non-human animal includes atleast a part of a human immunoglobulin gene. For example, it is possibleto engineer mouse strains deficient in mouse antibody production withlarge fragments of the human Ig loci. Using the hybridoma technology,antigen-specific monoclonal antibodies derived from the genes with thedesired specificity may be produced and selected. U.S. Pat. No.7,064,244.

Humanized antibodies may also be produced, for example, using transgenicmice that express human heavy and light chain genes, but are incapableof expressing the endogenous mouse immunoglobulin heavy and light chaingenes. Winter describes an exemplary CDR-grafting method that may beused to prepare the humanized antibodies described herein (U.S. Pat. No.5,225,539). All of the CDRs of a particular human antibody may bereplaced with at least a portion of a non-human CDR, or only some of theCDRs may be replaced with non-human CDRs. It is only necessary toreplace the number of CDRs required for binding of the humanizedantibody to a predetermined antigen.

Humanized antibodies or fragments thereof can be generated by replacingsequences of the Fv variable domain that are not directly involved inantigen binding with equivalent sequences from human Fv variabledomains. Exemplary methods for generating humanized antibodies orfragments thereof are provided by U.S. Pat. No. 5,585,089; U.S. Pat. No.5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 5,859,205; and U.S.Pat. No. 6,407,213. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable domains from at least one of a heavy or lightchain. Such nucleic acids may be obtained from a hybridoma producing anantibody against a predetermined target, as described above, as well asfrom other sources. The recombinant DNA encoding the humanized antibodymolecule can then be cloned into an appropriate expression vector.

In certain embodiments, a humanized antibody is optimized by theintroduction of conservative substitutions, consensus sequencesubstitutions, germline substitutions and/or backmutations. An antibodyor fragment thereof may also be modified by specific deletion of human Tcell epitopes or “deimmunization” by the methods disclosed in U.S. Pat.No. 7,125,689 and U.S. Pat. No. 7,264,806. Briefly, the heavy and lightchain variable domains of an antibody can be analyzed for peptides thatbind to MHC Class II; these peptides represent potential T-cellepitopes. For detection of potential T-cell epitopes, a computermodeling approach termed “peptide threading” can be applied, and inaddition a database of human MHC class II binding peptides can besearched for motifs present in the VH and VL sequences. These motifsbind to any of the 18 major MHC class II DR allotypes, and thusconstitute potential T cell epitopes. Potential T-cell epitopes detectedcan be eliminated by substituting small numbers of amino acid residuesin the variable domains, or preferably, by single amino acidsubstitutions. Typically, conservative substitutions are made. Often,but not exclusively, an amino acid common to a position in humangermline antibody sequences may be used. The V BASE directory provides acomprehensive directory of human immunoglobulin variable regionsequences. These sequences can be used as a source of human sequence,e.g., for framework regions and CDRs. Consensus human framework regionscan also be used, e.g., as described in U.S. Pat. No. 6,300,064.

Antibody mimetics or engineered affinity proteins are polypeptide basedtarget binding proteins that can specifically bind to targets but arenot specifically derived from antibody V_(H) and V_(L) sequences.Typically, a protein motif is recognized to be conserved among a numberof proteins. One can artificially create libraries of these polypeptideswith amino acid diversity and screen them for binding to targets throughphage, yeast, bacterial display systems, cell-free selections, andnon-display systems. See Gronwall & Stahl, J Biotechnology, 2009,140(3-4), 254-269, hereby incorporated by reference in its entirety.Antibody mimetics include affibody molecules, affilins, affitins,anticalins, avimers, darpins, fynomers, kunitz domain peptides, andmonobodies.

Affibody molecules are based on a protein domain derived fromstaphylococcal protein A (SPA). SPA protein domain denoted Z consists ofthree α-helices forming a bundle structure and binds the Fc portion ofhuman IgG1. A combinatorial library may be created by varying surfaceexposed residues involved in the native interaction with Fc. Affinityproteins can be isolated from the library by phage display selectiontechnology. See Orlova et al., Cancer Res., 2007, 67:2178-2186, herebyincorporated by reference in its entirety.

Monobodies, sometimes referred to as adnectins, are antibody mimicsbased on the scaffold of the fibronectin type III domain (FN3). SeeKoide et al., Methods Mol. Biol. 2007, 352: 95-109, hereby incorporatedby reference in its entirety. FN3 is a 10 kDa, β-sheet domain, thatresembles the V_(H) domain of an antibody with three distinct CDR-likeloops, but lack disulfide bonds. FN3 libraries with randomized loopshave successfully generated binders via phage display (M13 gene 3, gene8; T7), mRNA display, yeast display and yeast two-hybrid systems. SeeBloom & Calabro, Drug Discovery Today, 2009, 14(19-20):949-955, herebyincorporated by reference in its entirety.

Anticalins, sometimes referred to as lipocalins, are a group of proteinscharacterized by a structurally conserved rigid β-barrel structure andfour flexible loops. The variable loop structures form an entry to aligand-binding cavity. Several libraries have been constructed based onnatural human lipocalins, i.e., ApoD, NGAL, and Tlc. See Skerra, FEBSJ., 275 (2008), pp. 2677-2683, hereby incorporated by reference in itsentirety.

The ankyrin repeat (AR) protein is composed repeat domains consisting ofa β-turn followed by two α-helices. Natural ankyrin repeat proteinsnormally consist of four to six repeats. The ankyrin repeats form abasis for darpins (designed ankyrin repeat protein) which is a scaffoldcomprised of repeats of an artificial consensus ankyrin repeat domain.Combinatorial libraries have been created by randomizing residues in onerepeat domain. Different numbers of the generated repeat modules can beconnected together and flanked on each side by a capping repeat. Thedarpin libraries are typically denoted N×C, where N stands for theN-terminal capping unit, C stands for the C-terminal capping domain andx for the number of library repeat domains, typically between two tofour. See Zahnd et al., J. Mol. Biol., 2007, 369:1015-1028, herebyincorporated by reference in its entirety.

Aptamers refer to affinity binding molecules identified from randomproteins or nucleic acid libraries. Peptide aptamers have been selectedfrom random loop libraries displayed on TrxA. See Borghouts et al.,Expert Opin. Biol. Ther., 2005, 5:783-797, hereby incorporated byreference in its entirety. SELEX (“Systematic Evolution of Ligands byExponential Enrichment”) is a combinatorial chemistry technique forproducing oligonucleotides of either single-stranded DNA or RNA thatspecifically bind to a target. Standard details on generating nucleicacid aptamers can be found in U.S. Pat. No. 5,475,096, and U.S. Pat. No.5,270,163. The SELEX process provides a class of products which arereferred to as nucleic acid ligands or aptamers, which has the propertyof binding specifically to a desired target compound or molecule. EachSELEX-identified nucleic acid ligand is a specific ligand of a giventarget compound or molecule. The SELEX process is based on the fact thatnucleic acids have sufficient capacity for forming a variety of two- andthree-dimensional structures and sufficient chemical versatilityavailable within their monomers to act as ligands (form specific bindingpairs) with virtually any chemical compound, whether monomeric orpolymeric.

VI. Terms

As used herein, “subject” refers to any animal, preferably a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “combined with” when used to describe theadministration of particle conjugates and any additional treatment(s),e.g. anticancer agent, means that the additional treatment(s) may beadministered prior to, together with, or after the administration ofparticle conjugates, or a combination thereof.

The term “polynucleotide” refers to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The polynucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

The term “oligonucleotide” generally refers to a short length ofsingle-stranded polynucleotide chain usually less than 30 nucleotideslong, although it may also be used interchangeably with the term“polynucleotide.”

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should be understood to include either single- ordouble-stranded forms of nucleic acid, and, as equivalents, analogs ofeither RNA or DNA. Such nucleic acid analogs may be composed ofnucleotide analogs, and, as applicable to the embodiment beingdescribed, may be single-stranded (such as sense or antisense) ordouble-stranded polynucleotides.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, for the sequence “A-G-T,” is complementary to the sequence“T-C-A.” Complementarity may be “partial,” in which only some of thenucleic acids' bases are matched according to the base pairing rules.Or, there may be “complete” or “total” complementarity between thenucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The term “hybridization” refers to the pairing of complementary nucleicacids. Hybridization and the strength of hybridization (i.e., thestrength of the association between the nucleic acids) is impacted bysuch factors as the degree of complementary between the nucleic acids,stringency of the conditions involved, the T_(m) of the formed hybrid,and the G:C ratio within the nucleic acids. A single molecule thatcontains pairing of complementary nucleic acids within its structure issaid to be “self-hybridized.”

EXPERIMENTAL VII. Synthesis and Characterization of 10-23DNAzyme-Modified Gold Nanoparticles (AuNPs)

3′ thiol-modified oligonucleotides (4 μM) were reduced and then mixedwith AuNPs suspended at a final concentration of 8 nM. The solution wasthen stabilized with SDS and salted to 0.7 M NaCl over a period of 3hours with intermittent sonication. The oligonucleotide density of thepurified DzT₁₀NPs, where T₁₀ refers to the thiolated poly T spacerlinking the DNAzyme to the nanoparticle, was 160±10oligonucleotides/particle based on a fluorescence DNA quantification kit(Invitrogen). To verify particle integrity, TEM and UV-Vis analysis wereperformed before and after AuNP functionalization, and indicated thatthe particles remained dispersed following modification with DNAzymes.The catalytic activity of these particles was determined by measuringthe rate of hydrolysis of a diribonucleotide within a DNA substrate thatwas functionalized with a 5′ 6-fluorescein (FAME) and a 3′ black holeQuencher™ (BHQ™) (FIG. 1A).

VIII. Direct DNAzyme Conjugation to the Nanoparticle Surface and Impacton Catalytic Activity

Liu & Lu disclose gold nanoparticle/DNAzyme assembly. See Chem. Mater.,2004, 16, 3231-3238. The DNAzyme is hybridized to linker strands thatbridged the terminal sequences of DNA-functionalized nanoparticles, andtherefore the DNAzymes were separated from the gold core. Hybridizing alarge number of 8-17 DNAzymes onto the surface of DNA-modified AuNPaggregates significantly reduces the activity of the enzyme.

Experiments were performed in order to determine if the stericallycrowded oligonucleotide environment of an RNA-cleaving DzNP wouldinhibit catalysis. The reaction rate was measured using atemperature-controlled fluorometer, and reported in units of mol min⁻¹by using a fluorescence calibration curve for a 3′FAM6 modifiedoligonucleotide standard. FIG. 1B top shows representative reactionkinetics for DzT₁₀NPs and free DzT₁₀ (See FIG. 4 for sequences) undercertain conditions (25° C., 4.2 nM=[DzT₁₀] and [DzT₁₀NP], 1 μMsubstrate, 20 mM Tris pH=7.4, 300 mM NaCl, 50 mM Mg²⁺). The initial rateof the reaction was determined from the linear slope of the plot (t<80min). The k_(obs) for soluble DzT₁₀ and DzT₁₀NP were 0.017 and 0.095 molmin⁻¹, respectively. DzNP, rather than its individual sub-units, wascompared to a single DNAzyme molecule. The nanoparticle functions as anensemble entity inside living cells. For comparison, the individualenzymes subunits in DzNPs showed an activity of 0.002 mole min⁻¹ whenassuming a 33% hybridization efficiency. DzNPs are fairly stable underthese conditions, and particles that were used in catalysis reactionsfor 12 hr only showed a 20% decrease in activity (as measured by theinitial rate of reaction) when they were re-used the following day. Thisdata indicates that the DzNPs are robust and retain catalytic activitydespite the dense oligonucleotide environment on the surface of thenanoparticle.

IX. Assemblies are Sensitive to DNAzyme Density and Orientation

To study the effect of surface packing density and steric crowding onthe rate of catalysis, a series of DzNPs were synthesized using a binarymixture of two oligonucleotides that included a T₁₀ passivating sequencealong with the DzT₁₀ (FIG. 1A). In this series, the total ssDNAconcentration was kept constant while adjusting the molar concentrationsof 3′ thiol modified DzT₁₀ and T₁₀. Because both oligonucleotides havethe same T₁₀ spacer, the DNA composition of AuNPs was expected toreflect that of the bulk solution. The goal was to tune the averagespacing between adjacent DNAzymes and consequently tune steric crowding.The total number of DNA molecules per particle was measured using acommercial fluorescence assay (FIG. 1B middle). To further verify thesemeasurements, DNA was released from the particle surface usingdithiothreitol (DTT) and the DNAzyme concentration was determined byusing the observed rate constant of substrate hydrolysis as compared toa calibration standard of soluble DNAzyme.

FIG. 1B bottom shows a plot of the initial rate constant of DzNPparticles with a range of enzyme packing densities compared to solubleDNAzyme. At lower enzyme packing densities (15-60 enzymes/particle), theactivity of each particle shows a linear increase as a function of thenumber of DNAzymes per particle. However, particles that have packingdensities that exceed 60 DNAzymes per particle show saturation inactivity. This trend indicates that steric packing limits the maximumactivity of each DzNP assembly. As a corollary, this limit in maximumactivity equates to each catalytic oligonucleotide providing a footprintof greater than ˜11 nm² on the particle surface in order to achieve itsfull activity.

X. DNAzymes Catalytically Regulate Intracellular Gene Expression

Given that the hybridization efficiency and gene regulation efficacyhave been shown to be dependent on the chemical nature of the groupanchoring an oligonucleotide to an AuNP, the catalytic properties ofDzNPs modified with the following poly T and ethylene glycol phosphatelinkers: T₁₀, T₂₀, T₁₀N₄₀, and ((EG)₆PO₃)₃, were investigated. Themeasured oligonucleotide density for these fully packed particles was148, 137, 80, and 195 oligonucleotides/particle for the T₁₀, T₂₀,T₁₀N₄₀, and ((EG)₆PO₃)₃ linkers, respectively. It was expected thatlonger linkers would generate DzNPs with increased catalytic efficiency,as this difference would be due to reduced steric inhibition for longerlinkers. Surprisingly, no clear trend was observed for increasing linkerlength. In fact, DzT₁₀N₄₀NP (0.011 mol min⁻¹) had a slight decrease inactivity when compared to DzT₁₀NP (0.03 mol min⁻¹) and DzT₂₀NP (0.03 ofmol min⁻¹); whereas, DzT₁₀NP and DzT₂₀NP had identical activities. Theethylene glycol phosphate (EG)₆PO₃)₃ linker generated the most denselypacked particles (˜195 Dz molecules/AuNP), and when these particles werecompared to DzT₁₀NP and DzT₂₀NP, they showed a 56% increase in k_(obs).The results indicate that chemisorption of DNAzyme active sitenucleobases to the gold surface alters their catalytic activity, and mayplay a significant role in tuning the catalytic activity of DzNPs, inaddition to steric crowding (FIG. 1B).

The role of non-specific interactions in DzNP catalytic activity wasverified when the rate of substrate hydrolysis was measured using AuNPsfunctionalized with DNAzymes anchored through a 5′ thiol (Dz_(rev)T10).DNA density on these particles was 180 oligonucleotides/AuNP, but wefound that their activity was nearly abolished (FIG. 2A). Importantly,Dz_(rev)T₁₀NPs remained inactive even when their packing densities werereduced to 90 Dz/AuNP (˜50% packed) either with or without T₁₀passivation. Furthermore, when these particles are treated withmercaptoethanol, the Dz surface density is decreased, but again, theactivity of the Dz_(rev)T10NPs remained suppressed. When Dz_(rev)T₁₀NPswere treated with DTT in order to release the surface-bound DNA, thefree DzT₁₀ fully recovered its activity, thus displaying a 3200%increase in catalytic activity (FIG. 2B). The drastic difference inactivity between DzT₁₀NP and DzrevT₁₀NP (900% difference) indicates thatthe catalytic core is asymmetric in its sensitivity to the supportinggold nanoparticle surface. This observation agrees with systematicmutagenesis analysis studies on the 10-23 DNAzyme catalytic domain thathave shown a high degree of sensitivity at many bases near the 5′terminus of the active site. Therefore, DNAzyme orientation needs to becarefully examined in DzNP design.

XI. Photodynamic Release

Photodynamic control of gene regulation agents, DNAzymes specifically,is highly desirable, and various synthetic strategies have been employedto demonstrate this effect. Because the gold nanoparticle was found toinhibit the Dz anchored through its 5′ terminus, photo-induced DNArelease would be a suitable proof-of-concept to demonstrate thiscapability. Dz_(rev)T10 was released from the particle by irradiationwith a 532 nm pulsed laser, which is known to selectively cleave thethiol-gold bond at certain laser powers (FIG. 2C). Importantly, thisstrategy offer significant advantages because it is synthetically facileand compatible with 2-photon and visible irradiation, which is incontrast to the recently developed azo-benzene and caged-nucleotidebased approaches that require excitation at UV wavelengths.

XII. DzNPs are Superior as a Gene Regulation Agent

To test DzNP resistance towards nucleases, the catalytic activity offree DzT₁₀ and DzT₁₀NP was measured before and after incubation with amodel nuclease, DNase I. FIG. 3A shows that after DNase I treatment (120min), the soluble enzyme retained only 10% of its original activity,while the DzNPs retained 90% of its original activity. Having confirmedthe activity and nuclease resistance of DzNPs in vitro, experiments wereperformed to determine whether DzNPs can readily enter mammalian cellsand catalytically regulate gene expression. To test this, Dz_(GFP)NPswere designed with recognition arms specific towards the greenfluorescent protein (GFP) mRNA sequence expressed in a transientlytransfected model HeLa cell line. Two 2′ methyl ether linkages wereincorporated within both the 5′ and 3′ termini of the DNA sequence inorder to further reduce nuclease degradation. Flow cytometry was used toquantify the GFP expression levels on a per-cell basis in all samples.

To distinguish between antisense and DNAzyme-catalyzed hydrolysismechanisms, a single base mutation was introduced in the Dz catalyticcore (G1 to A1) to generate catalytically inactive nanoparticles(i-Dz_(GFP)NP). The oligonucleotide density was quantified and found tobe 83 and 99 DNAzymes per particle for i-Dz_(GFP)NP and Dz_(GFP)NP,respectively. HeLa cells were treated with (10 or 20) nM of Dz_(GFP)NPand i-Dz_(GFP)NP for 48 hrs, and then released from the cell cultureflask and analyzed using flow cytometry. The average fluorescenceintensity and standard deviation of triplicate wells are shown in FIG.3B. The data indicates that the enzymatic nanoparticles reduce GFPexpression in a dose-dependent manner for Dz_(GFP)NPs, whereasi-Dz_(GFP)NPs show little knockdown. Since both DNA-modifiednanoparticles are complementary to the GFP mRNA, they are expected toreduce GFP expression levels through an antisense knockdown mechanism.Therefore, the difference in their activity of 20% can be attributed tothe contributions of the DNAzyme catalytic core.

1. A particle conjugated to a catalytically cleaving nucleic acidwherein the nucleic acid comprises a sequence that cleaves a nucleicacid.
 2. The particle of claim 1, wherein the diameter of the particleis about 500 nm to 5 nm.
 3. The particle of claim 1, wherein particlecomprises gold, silver, iron, or iron oxide.
 4. The particle of claim 1,wherein the particle is conjugated to the nucleic acid through a linkinggroup comprising a thiol group, metal binding ligand, ethylene glycolpolymer, alkyl chain, ester group, or amide group.
 5. The particle ofclaim 1, wherein the particle is further conjugated to a targetingmoiety and/or siRNA.
 6. The particle of claim 1, wherein the cleavingnucleic acid comprises a sequence functioning to hybridize to a targetof interest.
 7. The particle of claim 1, wherein the cleaving nucleicacid is a DNAzyme or RNAzyme.
 8. The particle of claim 1, wherein thecleaving nucleic acid is 10-23 DNAzyme coating the particle at about onenucleic acid to about 3 to 12 square nanometers of the particle surface.9. A pharmaceutical composition comprising a particle of claim 1 and apharmaceutically acceptable excipient.
 10. The pharmaceuticalcomposition of claim 10 in the form of a buffered saline solution,capsule, pill, or tablet.
 11. A method of treating or preventing cancercomprising administering an effective amount of a pharmaceuticalcomposition of claim 9 to a subject in need thereof.
 12. The method ofclaim 11, wherein the cancer is selected from brain, lung, cervical,ovarian, colon, breast, gastric, skin, ovarian, pancreatic, prostate,neck, and renal cancer.
 13. The method of claim 11, wherein, thepharmaceutical composition is administered in combination with a secondanticancer agent.
 14. The method of claim 13 wherein, the secondanticancer agent may be selected from temozolamide, bevacizumab,procarbazine, lomustine, vincristine, gefitinib, erlotinib, docetaxel,cis-platin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed,methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin, vinblastine, vindesine, vinorelbine,taxol, taxotere, etoposide, teniposide, amsacrine, topotecan,camptothecin, bortezomib, anagrelide, tamoxifen, toremifene, raloxifene,droloxifene, iodoxyfene, fulvestrant, bicalutamide, flutamide,nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol,anastrozole, letrozole, vorazole, exemestane, finasteride, marimastat,trastuzumab, cetuximab, dasatinib, imatinib, combretastatin,thalidomide, and/or lenalidomide or combinations thereof.
 15. The methodof claim 14, wherein the subject is exposed to electromagnetic radiationunder conditions such that the administered particle releases nucleicacids conjugated to the particle.